1. Field of the Invention
The present invention relates to specific binding assays (e.g., immunoassays) which are useful for the determination of an analyte in a liquid test medium. In particular, the present invention relates to heterogeneous specific binding assays for the determination of the amount of suspected analyte in an aqueous test medium involving the formation and subsequent physical separation of "bound" and "free" forms of a labeled reagent from one another in order to complete the assay.
2. Description of the Prior Art
Heretofore, various binding assays using immobilized or immobilizable materials for the direct immobilization of one of the binding participants in a binding assay reaction, e.g., immobilized antigen or antibody, in order to accomplish the desired separation of the bound and free forms of a labeled reagent, have been proposed. In particular, a number of such binding assays have been described wherein an antibody to an antigen to be detected is bound to an immobilizing material such as the inner wall of a test tube or a plastic bead.
For example, in U.S. Pat. No. 4,243,749, a competitive binding assay is disclosed wherein a reaction is carried out in a test tube having a specific antibody to a hapten under determination insolubilized or immobilized on the inner wall of the test tube. The reaction includes a labeled hapten conjugate wherein the quantity of the labeled hapten conjugate which becomes bound to the test tube wall is inversely proportional to the amount of the hapten under determination.
Another of such binding assays is described by U.S. Pat. No. 4,230,683 which discloses a method employing a 6 mm polystyrene bead having antigen or antibody bound thereto wherein the antigen or antibody is reacted with a hapten-conjugated antibody to the antigen or antibody. The bound hapten-conjugated antibody is further reacted with labeled anti-hapten antibody in order to determine the amount of antigen or antibody in a test sample.
Still another of such binding assays is described by U.S. Pat. No. 4,228,237 which discloses a method for the detection and determination of ligands in a liquid medium using enzyme labeled avidin and a biotin labeled reagent in a specific binding process. In this method, the ligand to be detected is contacted with an insoluble phase containing a specific binding substance for the ligand.
In addition to the direct immobilization techniques heretofore described, indirect immobilization by marking or labeling a binding assay reaction participant to be immobilized with a first binding substance, and then adding an immobilized second binding substance, has been proposed.
For example, U.S. Pat. No. 4,298,685 discloses an enzyme immunoassay wherein a sample containing a biological substance under determination is mixed with antibodies to the biological substance tagged with biotin and with an enzyme-labeled form of the substance under assay. An immobilized form of avidin is then added wherein the avidin binds to the biotin-tagged antibody to immobilize the antibody-bound fraction of the enzyme-labeled reagent. Similarly, United Kingdom Patent Application No. GB 2,084,317A discloses an antigen-linked competitive enzyme immunoassay using avidin bound to a solid material and a biotin-labeled antigen.
Accordingly, it is an object of the present invention to provide a specific binding assay for the detection of an analyte in a liquid test medium that does not require centrifugation steps or similarly complex, cumbersome separation techniques as heretofore described to separate an immobilized complex from the reaction solution before the amount of analyte can be determined.
Further, it is an object of the present invention to provide an immobilizable component having more than one molecule of the binding partner under determination bound thereto so that a minimal amount of binding substance which precipitates the immobilizable component is needed.
Another object of the present invention is to provide an immobilizable component having an enhanced surface area or availability of a binding partner for a specific binding assay reaction.
It is still a further object of the present invention to provide a specific binding assay which can be performed within a reaction vessel without the need for additional rinsing or washing steps after the specific binding reaction has taken place and where only a small aliquot of a reaction solution is needed to determine the amount of analyte in the test medium.